This is a continuing project for which Mariana Dimitrova has made many new measurements including those with the inactive EI(H189A) in the presence of PEP, pyruvate, and Mg(II). The bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) couples the phosphorylation and translocation of specific sugars across the membrane. The activity of the first protein in this pathway, enzyme I (EI), is regulated by a monomer-dimer equilibrium where the Mg(II)-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI of the E. coli PTS as well as for mutants in which Glu or Ala is substituted for the active-site His189 [EI(H189E) or EI(H189A), respectively] have been measured under a variety of conditions by sedimentation equilibrium at pH 7.5, 4 and 20 C. Concurrently, thermal unfolding of these forms of EI have been monitored by differential scanning calorimetry and by changes in the intrinsic tryptophanyl residue fluorescence. Phosphorylated EI and EI(H189E) have 10-fold increased dimerization constants [log K = 6.3, expressed per M monomer] compared to those of dephospho-EI and EI(H189A) at 20 C. Dimerization is strongly promoted by 1 mM PEP with 2 mM Mg(II) [log K > 8 at 4 or 20 C], as demonstrated with EI(H189A) which cannot undergo autophosphorylation. Together, 1 mM PEP and 2 mM Mg(II) also markedly stabilize and couple the unfolding of C- and N-terminal domains of EI(H189A) - increasing the transition temperature (Tm) for unfolding the C-terminal domain by ca. 18 C and that for the N-terminal domain by ca. 9 C to Tm = 63 C, giving a dissociation constant of ca. 0.003 mM for the dissociation of PEP from the C-domain at 45 C. PEP alone also promotes the dimerization of EI(H189A), but only increases Tm ca. 5 C for C-terminal domain unfolding without affecting N-terminal domain unfolding, giving an estimated dissociation constant of 0.2 mM for PEP dissociation in the absence of Mg(II) at 45 C. In contrast, the presence of 5 mM pyruvate with 0.2-1.0 mM PEP and 2 mM Mg(II) decouples the unfolding of N- and C-terminal domains and promotes monomeric EI(H189A), indicating that intracellular concentrations of Mg(II), pyruvate, and PEP control the activity of enzyme I in the first step of the phosphotransferase system. Thus, the separation of substrate binding effects from those of phosphorylation by studies with wild-type EI and the inactive EI(H189A) has shown that intracellular concentrations of Mg(II), PEP, and pyruvate are important determinants of both conformational stability and dimerization of dephospho-enzyme I. In addition, the substrate analogue phosphonopyuvate (0. 65 mM) has been found to slowly bind to wild-type enzyme I in the presence of 2 mM Mg(II) and to slowly stabilize a conformation similar to that of EI(H189A) when incubated with 1 mM PEP and 2 mM Mg (II). This observation has a potential use in obtaining crystals of enzyme I suitable for x-ray structural analysis.